Defined mutations in the 5' nontranslated sequence of Sindbis virus RNA

Abstract

We have constructed 24 deletion mutants which contain deletions of from 1 to 15 nucleotides in the 5' nontranslated region of Sindbis virus RNA and tested the effect of these mutations on virus replication. The results showed that the first 44 nucleotides, which are capable of forming a hairpin structure, are important for virus replication, as all deletions tested in this region were either lethal or resulted in virus that grew poorly in comparison to the parental virus. Many of these deletions had different effects in mosquito cells than in chicken cells, suggesting that cellular factors, presumably proteins, bind to this region. This domain may function in at least two processes in viral replication. It seems likely that in the minus strand, this sequence element is bound by the viral replicase and promotes RNA replication. In the plus strand, this element may modulate initiation of translation of the nonstructural proteins. The results suggest that the hairpin structure itself is important. All deletions within it had deleterious effects on virus replication, and in particular, deletion of one of the G residues at nucleotide 7 or 8 or of one of the C residues at nucleotide 36 or 37 which are theoretically base-paired with these G's resulted in temperature-sensitive viruses that behaved very similarly. In contrast, large deletions between the 44-nucleotide hairpin and the translation start site at nucleotides 60 to 62 resulted in virus that grew as well as or better than the parental virus in both chicken and mosquito cells. The A residue at position 5 of the HRSP strain used was examined in more detail. Deletion of this A was lethal, whereas substitution by G resulted in a virus that grew poorly, despite the fact that G is present at position 5 in the AR339 parent of HRSP. U at position 5 resulted in a virus that grew less well than the A5 strain but better than the G5 mutant.